Currently used methods of stable plant transformation usually employ direct (microprojectile bombardment, electroporation or PEG-mediated treatment of protoplasts, for review see: Gelvin, S. B., 1998, Curr. Opin. Biotechnol., 9, 227-232; Hansen & Wright, 1999, Trends Plant Sci., 4, 226-231) or Agrobacterium-mediated delivery of pre-engineered DNA vectors of interest into plant cells. Manipulations with said DNA vectors in planta are restricted to simplifying the resolution of complex integration patterns (U.S. Pat. No. 6,114,600; Srivastava & Ow, 2001, Plant Mol Biol., 46, 561-566; Srivastava et al., 1999, Proc. Natl. Acad. Sci. USA, 96, 11117-11121) or removal of auxiliary DNA sequences from vectors stably integrated into chromosomal DNA. The methods of stable Agrobacterium-mediated integration of T-DNA regions within plant cells use a desired DNA sequence to be integrated flanked with left (LB) and right (RB) border sequences necessary for T-DNA transfer and integration into the host chromosomal DNA (U.S. Pat. Nos. 4,940,838; 5,464,763; EP0224287; U.S. Pat. Nos. 6,051,757; 5,731,179; WO9400977; EP0672752). In most cases the approaches are directed to integration of one specific T-DNA region into the chromosomal DNA, less frequently the approaches are designed for co-integration of two or more different T-DNA regions (U.S. Pat. No. 4,658,082). The latter approach is used for segregating different T-DNAs in progeny for various purposes. For example, Komari and colleagues (U.S. Pat. No. 5,731,179) describe a method of simultaneously transforming plant cells with two T-DNAs, one carrying a selectable marker functional in plants, while another T-DNA contains a desired DNA sequence to be introduced into plant cells. This allows to segregate in progeny transgenic plants without selectable marker.
The integration of a gene of interest into chromosomal DNA for expressing said gene can also be performed with the help of vectors that do not contain functional transcriptional promoters, but translation regulatory elements (WO0246440) called IRESs (internal ribosomal entry sites). Such vectors can provide for the expression of the gene of interest upon integration into the transcriptionally active region of chromosomal DNA. Another approach to provide for the expression of a promoterless gene or gene with minimal promoter also depends on integration into transcriptionally active regions (Stangeland et al., 2003, J. Exp. Bot., 54, 279-290; Baxter-Burrell et al., 2003, Ann. Bot. (Lond), 91, 129-141) or in close proximity to strong transcriptional enhancers (Baxter-Burrell et al., 2003, Ann. Bot. (Lond), 91, 129-141).
In general, the DNA regions designed for stable integration into plant cells are pre-engineered in vitro by employing standard molecular biology techniques (Sambrook, Fritsch & Maniatis, 1989, Molecular cloning: A laboratory manual, 2nd ed. Cold Spring Harbor, N.Y.: CSH Laboratory Press). Also, in vivo engineering in bacterial cells is used, for example in order to assemble a binary vector with the help of homologous recombination (U.S. Pat. No. 5,731,179). Manipulations with T-DNA in planta are restricted to T-DNA regions pre-integrated into a chromosome. Such manipulations were done for removing certain sequences from T-DNA, e.g. sequences encoding selectable markers including morphological abnormality induction genes. The removal of unwanted DNA fragments from T-DNA regions was tried either with the help of site-specific recombination (WO9742334; Endo et al., 2002, Plant J., 30, 115-122) or by means of transposition (U.S. Pat. No. 5,792,924). Although site-specific recombinase/integrase-mediated DNA excision is more efficient than integration, the selection for excision events is a necessity, which leads to yet an additional step of tissue culture or screening of progeny for desired recombination events. In summary, all processes of manipulation with T-DNAs stably integrated into plant chromosomes are time-consuming, inflexible, and in general restricted to simple excision (with less efficiency—to integration) of desired DNA fragments. In addition, these processes are usually very limited in combinatorial diversity, as they are restricted to simple manipulations with a limited number of known genes and regulatory elements.
Frequently, T-DNA regions co-integrate into the same locus (Jorgensen et al., 1987, Mol. Gen. Genet., 207, 471-477; Castle et al., 1993, Mol. Gen. Genet., 241, 504-514; Cluster et al, 1996, Plant Mol. Biol., 32, 1197-1203; DeNeve et al., 1997, Plant J., 11, 15-29) forming multimers of T-DNA regions. However, such complex integration events are usually undesired in any respect, as such complex arrangements are accompanied by the inactivation of the transgene (Cluster et al., 1996, Plant Mol. Biol, 32, 1197-1203; Jorgensen et al., 1996, Plant Mol. Biol., 31, 957-973). Transformation of plants with two different T-DNA regions, one carrying a coding sequence of a transformation marker, was used for generating transformation marker-free transgenic plants (Komari et al., 1996, Plant J., 10, 165-174). Co-integration of two copies of T-DNAs, one carrying a promoter and another carrying a promoterless neomycin phosphotransferase gene, was used to study the T-DNA cointegration pattern (Krizkova & Hrouda, 1998, Plant J., 16, 673-680).
The methods described above suffer from various shortcomings. In the method of Krizkova & Hrouda, one does not select for cointegration of both vectors (or both copies of the same vector). Instead, expressible transformation of the neomycin phosphotransferase gene is selected for, which may be due to fortuitous insertion of the promoterless neomycin phosphotransferase gene in a transcriptionally active chromosome region. Further, in methods using a single transformation vector, complex and time-consuming cloning procedures are required e.g. if the plant cells are to be transformed with a complex combination of sequences of interest (e.g. more than one gene to be expressed together with specific elements like recombination sites, regulatory sequences, transposon sequences etc.). Moreover, the above methods are not suited for methods of in vivo engineering like gene (or protein domain) shuffling or directed evolution. Furthermore, the above methods do not allow to obtain biologically safe transgenic plants, whereby the transgene of said transgenic plants is lost or rendered disfunctional in progeny of said transgenic plants.
Therefore, it is an object of the invention to provide an efficient, rapid and highly versatile process of producing a transgenic plant or transgenic plant cells. It is another object of the invention to provide a method of selecting for co-integration of two or more vectors transformed in plant cells. It is another object of the invention to provide a process of producing transgenic plants transformed on a chromosome, whereby the DNA sequences of interest are to be engineered in planta (e.g. for reducing cloning work or for transforming a DNA sequence of interest having toxic effects on bacteria that used for cloning). It is another object of the invention to provide a process of stable genetic transformation of plant cells, which allows the production of a library of traits or functions and/or screening for a desired trait (or function) from a library of traits (or functions). It is a further object of the invention to provide a process of producing environmentally safe transgenic plants, that transfer the transgenic function or trait to progeny with a low likelihood.